Production of vaccines

ABSTRACT

Means and methods for producing mammalian viruses, the method comprising infecting a culture of immortalized human cells with a virus, incubating the culture infected with virus to propagate the virus under conditions that permit growth of the virus, and to form a virus-containing medium, and removing the virus-containing medium. The viruses can be harvested and be used for the production of vaccines. Advantages include that human cells of the present invention can be cultured under defined serum-free conditions and the cells show improved capability for propagating virus. Methods are provided for producing, in cultured human cells, influenza virus and vaccines derived thereof. This method eliminates the necessity of using whole chicken embryos for the production of Influenza vaccines. The method also provides for the continuous or batch-wise removal of culture media. As such, the present invention allows the large-scale continuous production of viruses to a high titer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/722,867, filed Nov. 27, 2000, pending, which is acontinuation-in-part of U.S. patent application Ser. No. 09/449,854filed on Nov. 26,1999, the entire contents of each of which areincorporated by this reference.

TECHNICAL FIELD

This invention relates generally to biotechnology, and more particularlyto the development and manufacture of vaccines. In particular, theinvention relates to the production of viral proteins and/or virusesusing a mammalian (e.g., human) cell for the production of virusesgrowing in eukaryotic, especially mammalian and human, cells. Theinvention is useful for the production of vaccines to aid in protectionagainst viral pathogens for vertebrates, such as mammals.

BACKGROUND

Presently, vaccination is the most important route of dealing with viralinfections. Although a number of antiviral agents are available,typically, these agents have limited efficacy. Administering antibodiesagainst a virus may be a good way of dealing with viral infections oncean individual is infected (passive immunization). Typically, human orhumanized antibodies hold promise for dealing with a number of viralinfections, but the most efficacious and safe way of dealing with virusinfection presently is, and probably will be, prophylaxis through activeimmunizations. Active immunization is generally referred to as“vaccination.” Vaccines comprise at least one antigenic determinant(typically of a virus), preferably a number of different antigenicdeterminants of at least one virus or other pathogen, for instance, byincorporating in the vaccine at least one (viral) polypeptide or proteinderived from a virus (subunit vaccines).

Typically, vaccines include adjuvants in order to enhance the immuneresponse. Use of adjuvants is also possible for vaccines that use wholevirus (pathogen), for instance, when the virus is inactivated. Anotherpossibility is the use of live, but attenuated, virus. A furtherpossibility is the use of wild-type (“wt”) virus, for instance, in caseswhere adult individuals are not in danger of infection but infants areand may be protected through maternal antibodies and the like.

Producing vaccines is not always an easy procedure. In some cases, theproduction of viral material is on eggs, which may lead to materialsthat are difficult to purify and require extensive safety measuresagainst, for instance, contamination. Likewise, production on bacteriaor yeast, which is sometimes an alternative for eggs, can require manypurification and safety steps.

Production on mammalian cells would be an alternative, but the mammaliancells used thus far have required, for instance, the presence of serumand/or adherence to a solid support for growth. In the first case, againpurification and safety and, for example, the requirement of protease tosupport the replication of some viruses, becomes an issue. In the secondcase, high yields and ease of production become a further issue. Thepresent invention overcomes at least a number of the problemsencountered with the production systems for production of viruses and/orviral proteins for vaccine purposes of the systems of the prior art.

BRIEF SUMMARY OF THE INVENTION

The invention includes a novel human immortalized cell line for thepurpose of propagating, harvesting and producing virus. PER.C6 cells(see, e.g., U.S. Pat. No. 5,994,128 to Bout et al., also deposited underNo. 96022940 at the European Collection of Animal Cell Cultures at theCentre for Applied Microbiology and Research) were generated bytransfection of primary human embryonic retina cells using a plasmidthat contained the adenovirus (“Ad”) serotype 5 (AdS) E1A- andE1B-coding sequences (Ad5 nucleotides 459-3510) (SEQ ID NO:1) under thecontrol of the human phosphoglycerate kinase (PGK) promoter.

The following features make PER.C6 or a derivative thereof particularlyuseful as a host for virus production: it is a fully characterized humancell line; it was developed in compliance with good laboratorypractices; it can be grown as a suspension culture in defined serum-freemedium, devoid of any human or animal serum proteins; its growth iscompatible with roller bottles, shaker flasks, spinner flasks andbioreactors, with doubling times of about 35 hours.

Influenza Epidemiology

Influenza viruses, members of the family of Orthomyxoviridae, are thecausative agents of annual epidemics of acute respiratory disease. Inthe U.S. alone, 50 million Americans get the flu each year. Estimateddeaths worldwide (1972-1992) are 60,000 (CDC statistics). There havebeen three major cases of pandemics of influenza, namely in 1918(Spanish flu, estimated 40 million deaths), in 1957 (Asian flu,estimated 1 million deaths), and in 1968 (Hong-Kong flu, estimated700,000 deaths).

Infections with influenza viruses are associated with a broad spectrumof illnesses and complications that result in substantial worldwidemorbidity and mortality, especially in older people and patients withchronic illness. Vaccination against influenza is most effective inpreventing the often fatal complications associated with this infection(Murphy, B. R. and R. G. Webster 1996). The production of influenzavirus on the diploid human cell line MRC-5 has been reported(Herrero-Euribe L. et al. 1983). However, the titers of influenza viruswere prohibitively low.

Strains of Influenza Virus

Present day flu vaccines contain purified hemagglutinin andneuraminidase of Influenza virus A and B. The three viruses thatrepresent epidemiologically important strains are Influenza A (HIN1),Influenza A (H3N2) and Influenza B. The division into A and B types isbased on antigenic differences between their nucleoprotein (NP) andmatrix (M) protein antigen. The Influenza A virus is further subdividedinto subtypes based on the antigenic composition (sequence) ofhemagglutinin (H1-H15) and neuraminidase (N1-N9) molecules.Representatives of each of these subtypes have been isolated fromaquatic birds, which probably are the primordial reservoir of allinfluenza viruses for avian and mammalian species. Transmission has beenshown between pigs and humans and, recently (H5N1), between birds andhumans.

Influenza Vaccines

Three types of inactivated influenza vaccine are currently used in theworld: whole virus, split product, and surface antigen or “subunit”vaccines. These vaccines all contain the surface glycoproteins,hemagglutinin (HA) and neuraminidase (NA) of the influenza virus strainsthat are expected to circulate in the human population in the upcomingseason. These strains, which are incorporated into the vaccine, aregrown in embryonated hens' eggs and the viral particles are subsequentlypurified before further processing.

The need for the yearly adjustment of influenza vaccines is due toantigen variation caused by processes known as “antigenic drift” and“antigenic shift.”

“Antigenic drift” occurs by the accumulation of a series of pointmutations in either the H or N protein of a virus resulting in aminoacid substitutions. These substitutions prevent the binding ofneutralizing antibodies induced by previous infection and the newvariant can infect the host.

“Antigenic shift” is the appearance of a new subtype by geneticre-assortment between animal and human Influenza A viruses. The pandemicstrains of 1957 (H2N2) and 1968 (H3N2) are examples of re-assortedviruses by which avian H and/or N genes were introduced in circulatinghuman viruses that subsequently spread among the human population.

Based on the epidemiological surveys by over one hundred NationalInfluenza Centres worldwide, the World Health Organization (WHO) yearlyrecommends the composition of the influenza vaccine, usually in Februaryfor the northern hemisphere and in September for the southernhemisphere. This practice limits the time window for production andstandardization of the vaccine to a maximum of nine months.

If an urgent demand arises for many doses of vaccine, for example, whena novel subtype of Influenza A virus arises by antigenic shift orantigenic drift, limited availability of eggs may hamper the rapidproduction of vaccine. Further disadvantages of this production systemare the lack of flexibility, the risk of the presence of toxins, and therisks of adventitious viruses, particularly retroviruses, and concernsabout sterility. These disadvantages present a serious problem intoday's practice of influenza vaccine production on embryonated hens'eggs.

Therefore, the use of a cell culture system for influenza vaccineproduction would be an attractive alternative. Influenza viruses can begrown on a number of primary cells, including monkey kidney, calfkidney, hamster kidney and chicken kidney. Yet, their use for vaccineproduction is impractical, due to the need to re-establish cultures fromthese primary cells for each preparation of a vaccine. Therefore, theuse of continuous immortalized cell lines for influenza vaccineproduction is an attractive alternative.

The use of culture systems was facilitated by the realization that theproteolytic cleavage of HA into its two subunits (HA1 and HA2) isrequired for influenza virus infectivity and can be obtained by addingtrypsin. Including trypsin permits replication and plaque formation inMadin-Darby canine kidney (MDCK) cells (Tobita et al. 1975).

The MDCK cell line was recently shown to support the growth of influenzavirus for vaccine production (Brand et al. 1996 and 1997, Palache et al.1997). The use of trypsin requires growth of the MDCK cells inserum-free tissue culture medium (MDCK-SF1). However, MDCK cells arecurrently not approved as a substrate for production of influenza virus.

Importantly, any non-human system for producing influenza vaccines hasan inherent drawback, known as “adaptation.” Human Influenza A and Bviruses both carry mutations in HA, due to adaptation in embryonatedhens' eggs. These mutations result in altered antigenicity (Newman etal. 1993, Williams and Robertson 1993, Robertson et al. 1994, Gubarevaet al. 1994, Schild et al. 1993, Robertson et al. 1987, Kodihalli et al.1995). In humans, immunization with vaccines containing HA bearing anegg-adaptation mutation induces less neutralizing antibody to virus thana non-egg adapted HA (Newman et al. 1993).

Human influenza viruses propagated in canine cells, such as MDCK cells,also show adaptation, albeit to a lesser extent. Such viruses resemblethe original human isolates more closely than egg-derived viruses(Robertson et al. 1990).

Furthermore, evidence exists that host-specific changes in NA andhost-specific phosphorylation patterns of NA can affect the replicationof Influenza viruses (Schulman and Palese 1977; Sugiara and Ueda 1980;Kistner et al. 1976).

Therefore, it would clearly be advantageous to avoid adaptation or otherhost-induced changes of influenza virus, possibly resulting in a morehomogeneous population of viruses, rendering the ultimate vaccine moreeffective.

The present invention provides human cells used as a substrate for theproduction of high titers of influenza virus suitable for thedevelopment of vaccines.

Rotavirus and Vaccines Therefor

Rotaviruses are the most important cause of severe dehydratinggastroenteritis in young children worldwide. In developing countries,infections with rotaviruses reportedly lead to over 800,000 deathsannually. In the United States alone, estimated costs of health care dueto rotavirus infections exceed 1 billion U.S. dollars per year.

Rotaviruses, members of the family of Reoviridae, are double strandedRNA viruses consisting of eleven RNA segments, each coding for astructural or non-structural viral protein (VP). This inner core of thevirus comprises four VPs: VP1, 2, 3 and 6. These VP determine the threemain antigenic properties of HRV—group, subgroup, and serotype. Sevenantigenically distinct groups (denominated A through G) have beenidentified that are encoded by the VP6. Infection with human rotavirus(HRV) is predominantly caused by group A rotaviruses, with serotypes 1-4accounting for 95% of clinical illness. Natural disease protection isserotype-specific. Group A is further classified into subgroups I andII.

The double layer outer shell forming the viral capsid consists of twoviral proteins, VP4 and VP7, that are the neutralization antigensinvolved in protective immunity and that determine the serotype,although the VP4 plays a minor role in serotype determination. Duringco-infection with different serotypes, the segmented genomes readilyundergo genetic re-assorting, a property that has been used to create avaccine (Marsha et al. 1999).

Given the worldwide prevalence of rotavirus-associated infant morbidityand mortality, large scale vaccination against rotavirus is consideredthe most effective way to combat this virus. The goal of vaccinationwould not be to prevent the disease but to reduce its morbidity,especially during the first few years of life.

The only effective vaccine available at present is a live, attenuated,orally delivered vaccine based on the re-assortment of RNA segments ofhuman rotaviruses, encoding the VP7s of serotypes 1, 2 and 4 in a Rhesusrotavirus supplying the attenuated background together with the VP7 ofserotype 3. Vaccination with this human/rhesus reassortant tetravalentvaccine (RRV-TV), although highly effective in preventing severegastroenteritis, is associated with intussusception, a bowel obstructiondisease. For that reason, this vaccine is no longer in use.

Means and methods are disclosed herein for producing a virus and/orviral protein in a cell, preferably using a defined synthetic medium,and for purifying the virus and/or components thereof from the celland/or culture medium. Pharmaceutical compositions containing virus orits components and methods for manufacturing and recovering and/orpurifying them are provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph depicting percentage of infected cells (positivecells) viewed microscopically after immunofluorescence assay versuspercentage of dead cells measured via FACS after propidium iodidestaining, at multiplicities of infection (mois) of 10⁻³ and 10⁻⁴. Poorviability of the cells from samples derived from infection at moi 10⁻³did not give rise to reliable data.

FIG. 2 consists of two graphs depicting percentage of infected cellsviewed microscopically after immunofluorescence assay. Samples derivedfrom infection at moi 10 and 1, at 48 hours post-infection are notshown, because of full CPE.

FIG. 3 is a graph depicting kinetics of virus propagation measured inhemagglutinating units (HAU) from day 1 through day 6 after infection.

FIG. 4 consists of two graphs depicting percentage of infected cells(positive cells) viewed microscopically after immunofluorescence assay.

FIG. 5 consists of two graphs depicting kinetics of virus propagationmeasured in HAU from day 1 through day 6 after infection.

FIG. 6 consists of two graphs depicting percentage of infected cells(positive cells) viewed microscopically after immunofluorescence assay.

FIG. 7 consists of two graphs depicting kinetics of virus propagationmeasured in HAU from day 2 through day 6 after infection.

FIG. 8 consists of portions A and B depicting expression of Sia2-3Galand Sia2-6Gal linkages on cell surface receptors present on ChineseHamster Ovary (CHO) cells, PER.C6 cells and MDCK cells. Portion A is aschematic representation of the interaction of the Sambucus nigraagglutinin (SNA) lectin that specifically recognizes Sia2-6Gal linkagesand the Maackia amurensis agglutinin (MAA) lectin that specificallyrecognizes Sia2-3Gal linkages. The schematic interaction with theFITC-labeled anti-DIG antibody recognizing the DIG-labeled lectin boundto the oligosaccharide chain on the cell surface protein is alsodepicted. Portion B depicts FACS analysis of cells incubated withDIG-labeled lectins. Lectins attached to the cells were detected withFITC-labeled anti-DIG antibody using procedures known to persons skilledin the art. Cell number counts are plotted against the fluorescenceintensity of lectin-stained cells (gray) as compared with cells thatwere incubated only with the FITC-anti-DIG antibody (open). The upperpanels of Portion B show the shift in the FACS analysis obtained byusing the SNA lectin, while the lower panels of Portion B show the shiftin the FACS analysis obtained by using the MAA lectin.

FIG. 9 consists of Portions A, B, and C, depicting infection withA/Sydney/5/97 on PER.C6. (A) Effect of trypsin-EDTA on HAU titers. (B)HA concentration in μg/ml and (C) virus infectivity titers in pfu/ml asmeasured in crude viral supernatants, 96 hours post-infection.

FIG.10 consists of Portions A, B, and C depicting infection withB/Harbin/7/94 on PER.C6. (A) Effect of different concentrations oftrypsin-EDTA present during and after virus infection on growthkinetics. (B) HAU titers per 50 μl and (C) virus infectivity titers inpfu/ml.

FIG. 11 consists of Portions A and B which depict infection with X-127using an moi of 10⁻³ on PER.C6. (A) Effect of trypsin-EDTA on HAU givenin HAU/50 μl and (B) virus infectivity titers in pfu/ml for five daysafter infection.

FIG. 12 consists of Portions A and B depicting infection with X-127using an moi of 10⁻⁴ on PER.C6. (A) Effect of trypsin-EDTA on HAU givenin HAU/50 μl and (B) virus infectivity titers in pfu/ml during five daysafter infection.

FIG. 13 consists of Portions A and B depicting effect of trypsin-EDTA on(A) PER.C6 cells viability and (B) biological activity of the virus.Cell viability was measured after trypan-blue staining. HAU titers weremeasured as described and given per 50 μl.

FIG. 14 consists of Portions A and B depicting effect of trypsin-EDTA onvirus infectivity titers and HA protein content after influenzainfection of PER.C6 cells with A/Sydney/5/97. (A) The infectivity assaywas carried out by inoculating, in quadruplicate, MDCK cells with atotal of 100 μl of ten-fold serially diluted virus-containingsupernatants, in serum-free medium with trypsin-EDTA (4 μg/ml). Afterseven days, supernatant of these cultures were tested for HA activity.The infectious virus titers were calculated according to the method ofSpearnan-Karber (1931). (B) Western blot analysis of the A/Sydney/5/97HA protein. Harvesting of viral proteins was carried out by disruptionand denaturation of proteins using an SDS-containing lysis buffer. Theelectrophoretic run was performed on a 10% SDS/PAGE gel under reducingconditions. Separated proteins were probed with the specificanti-A/Sydney-HA antisera. Increasing amounts of the positive controlA/Sydney HA antigen (left four lanes) and 10 μl of PER.C6 cellssupernatants of the indicated trypsin incubated samples (right fivelanes) were loaded.

FIG. 15 consists of an upper and lower portion and depict PER.C6 cellsviability, glucose concentration, and growth kinetics of A/Sydney/5/97in a hollow fiber perfusion system.

FIG. 16 consists of a right and left portion and depicts thecharacterization and quantification of Influenza Virus A/Sydney/5/97propagated on PER.C6 in a hollow fiber perfusion system. SDS-PAGE andWestern blots were done as described in legend to FIG. 14 for the Sheepanti-A/Sydney-HA antibody. The monoclonal antibody anti-HA-tag (HA probeF7) mouse monoclonal (Santa Cruz) was used in 1:1000 dilution. As asecond antibody, a goat anti-mouse-HRP-conjugated antibody (Biorad), in1:7500 dilution was used.

FIG. 17 consists of a right and left graph and depicts PER.C6 cellsviability (left panel) and glucose concentration (right panel) in a 12liter bioreactor up to 92 hours after viral infection usingA/Sydney/5/97 virus.

FIG. 18 is a graph depicting the infection of PER.C6 with A/Sydney/5/97in a 10 liter cell suspension in a 12 liter bioreactor. Kinetics ofvirus replication as measured by immunofluorescence assay are given inpercentages of positively stained cells.

FIG. 19 is a bar graph depicting infection of PER.C6 cells withA/Sydney/5/97 in a 10 liter cell suspension in a 12 liter bioreactor.Kinetics of virus replication as measured by Hemagglutination assay aregiven in HAUs during several days after viral infection. The bardepicted with an asterisk is the number of HAUs obtained afterPowerfuge™ clarification as described in the text.

FIG. 20 is a Western blot following infection of PER.C6 withA/Sydney/5/97 virus in a 10 liter cell suspension in a 12 literbioreactor. Shown is the characterization and quantification of theInfluenza virus A/Sydney/5/97 HA polypeptide. SDS/PAGE and Western blotwere done as described with respect to FIG. 14. The different subunits(HA1 and HA2) and the non-cleaved HA0 proteins are depicted by arrowheads. The HA obtained from NIBSC served as a positive control.

FIG. 21 shows the determination of HAUs and pfu/ml after infection ofPER.C6 with A/Sydney/5/97 in a 10 liter cell suspension in a 12 literbioreactor. The infection was followed by Down Stream Processing (DSP).The recovery of viral yields after hollow fiber ultra-filtration(20-fold concentration) is also shown.

FIG. 22 consists of four graphs depicting infection of PER.C6 withA/Sydney/5/97 in a 2 liter cell suspension in a 3 liter bioreactor.PER.C6 cells viability (upper left), glucose concentration (upper right)and growth kinetics of the virus in the percentage of positivelystaining cells (lower left), and HAUs (lower right) are given.

FIG. 23 consists of four graphs depicting infection of PER.C6 withA/Beijing/262/95 in a 2 liter cell suspension in a 3 liter bioreactor.PER.C6 cells viability (upper left), glucose concentration (upper right)and growth kinetics of the virus in the percentage of positivelystaining cells (lower left), and HAUs (lower right) are given.

FIG. 24 consists of four graphs depicting infection of PER.C6 withB/Harbin/7/94 in a 2 liter cell suspension in a 3 liter bioreactor.PER.C6 cells viability (upper left), glucose concentration (upper right)and growth kinetics of the virus in the percentage of positivelystaining cells (lower left), and HAUs (lower right) are given.

FIG. 25 is a Western blot analysis of uncleaved A/Sydney/5/97 HA0protein. Positive staining proteins are detected after incubation withthe specific anti-A/Sydney antisera obtained from NIBSC and described asin the legend of FIG. 14 and in the text.

FIG. 26A is a Western blot analysis of A/Sydney/5/97-derived HA0 proteindigested with trypsin. Proteins are detected after incubation with thespecific anti-A/Sydney antisera. On the left, a standard cleavedA/Sydney HA and on the right, HA0 treated with increasing amount oftrypsin.

FIG. 26B is a determination of trypsin activity in the culturesupernatant of an Influenza B/Harbin production run, using HAO ofInfluenza A/Sydney/5/97 as substrate. Western blot analysis of HA0cleavage products HA1 and HA2 as visualized by anti-InfluenzaA/Sydney/5/97 HA-specific antisera is mentioned in legend to FIG. 14.

FIG. 27 is a Western blot analysis of A/Sydney HA0 digested withN-glycosydase F. Proteins are detected after incubation with thespecific anti-A/Sydney antisera. The protein band depicted with anasterisk is the de-glycosylated product.

FIG. 28 is a Western blot analysis of A/Sydney/5/97 HA after Accutasedigestion. Proteins are detected after incubation with the specificpolyclonal anti-A/Sydney-HA antisera. On the left, HA0 before and aftertrypsin treatment, on the right, HA0 digested with decreasing amount ofAccutase.

FIG. 29 consists of five portions (A through E) and depicts electronmicrographs of Influenza A/Sydney/5/97. (A) PER.C6 cells 72 hourspost-infection. (B and C) Negative staining on virus derived frominfected PER.C6. (D and E) Negative staining of sucrose purifiedmaterial.

FIG. 30A identifies different Influenza A and B strains tested on PER.C6cells.

FIG. 30B is a bar graph depicting infectivity titers of three depictedA- and B-type influenza viruses derived from infected PER.C6 cells.

FIG. 31 consists of five bar graphs (A through E) depictingimmunofluorescence of PER.C6 and Vero cells infected with viruses otherthan influenza. (A) Positively staining cells upon infection withMeasles virus. (B) Positively staining cells upon infection of Verocells with HSV-1 virus. (C) Positively staining cells upon infection ofVero cells with HSV-2 virus. (D) Positively staining cells uponinfection of PER.C6 cells with HSV-1 virus. (E) Positively stainingcells upon infection of PER.C6 cells with HSV-2 virus.

FIG. 32 consists of upper, middle and lower portions, depictinginfectivity titers determined after propagation of measles virus (middlepanel), HSV-1 (bottom panel) and HSV-2 (top panel) virus on PER.C6cells.

FIG. 33 consists of upper and lower panels and depicts replication ofrotavirus after infection of PER.C6 (top panel) and Vero (bottom panel)cells with different mois as measured by ELISA in crude supernatants.

FIG. 34 is a graph depicting data from Example 19, showing that H5N1 andH9N2 infected almost all cells, while H7N7 was only detected in 10% ofthe cells.

FIG. 35 includes two graphs also depicting data from Example 19, showingthat the E1-transformed cells are able to sustain growth of all threetested influenza strains that are associated with a pandemic outbreak.

FIG. 36 includes a graph depicting further data from Example 19, showingthat at even at very low inoculation titers, good results can beachieved.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for producing a virus and/or viralproteins, other than adenovirus or adenoviral proteins, for use as avaccine comprising: providing a cell with at least a sequence encodingat least one gene product of the E1 gene, or a functional derivativethereof, of an adenovirus; providing the cell with a nucleic acidencoding the virus or the viral proteins; culturing the cell in asuitable medium and allowing for propagation of the virus or expressionofthe viral proteins; and harvesting the virus and/or viral proteinsfrom the medium and/or the cell.

Heretofore, few (if any) human cells have been found that were suitableto produce viruses and/or viral proteins for use as vaccines in anyreproducible and scalable manner, in sufficiently high yields, and/oreasily purifiable. We have now found that cells having adenoviral E1sequences (preferably in their genome) are capable of sustaining thepropagation of viruses in significant amounts.

A preferred cell according to the invention is derived from a humanprimary cell, preferably a cell which is immortalized by a gene productof the E1 gene. In order to be able to grow a primary cell, it, ofcourse, needs to be immortalized. A good example of such a cell is onederived from a human embryonic retinoblast.

In cells according to the invention, it is important that the E1 genesequences are not lost during the cell cycle. It is, therefore,preferred that the sequence encoding at least one gene product of the E1gene is present in the genome of the human cell.

For safety reasons, care is best taken to avoid unnecessary adenoviralsequences in the cells. It is thus another embodiment of the inventionto provide cells that do not produce adenoviral structural proteins.However, in order to achieve large scale (continuous) virus productionthrough cell culture, it is preferred to have cells capable of growingwithout needing anchorage. Preferred cells according to the inventionhave that capability. To have a clean and relatively safe productionsystem from which it is easy to recover and, if desired, purify thevirus, it is preferred to have a method according to the inventionwherein the human cell comprises no other adenoviral sequences. The mostpreferred cell for the methods and uses of the invention is thepreviously identified PER.C6 cell or a derivative thereof.

Thus, the invention provides a method of using a cell, wherein the cellfurther comprises a sequence encoding E2A or a functional derivative,analogue or fragment thereof, preferably a cell wherein the sequenceencoding E2A or a functional derivative, analogue or fragment thereof ispresent in the genome of the human cell and, most preferably, a cellwherein the E2A encoding sequence encodes a temperature-sensitive (ts)mutant E2A.

Furthermore, as previously stated, the invention also provides a methodwherein the human cell is capable of growing in suspension.

The invention also provides a method wherein the human cell can becultured in the absence of serum. A cell according to the invention, inparticular PER.C6, preferably has the additional advantage that it canbe cultured in the absence of serum or serum components. Thus, isolationis easy, safety is enhanced, and the system has good reliability(synthetic media are the best for reproducibility). The human cells ofthe invention and, in particular, those based on primary cells,particularly ones based on HER cells, are capable of normal (for humans)post- and peri-translational modifications and assembly. This means thatthey are very suitable for preparing viral proteins and viruses for usein vaccines.

Thus, the invention provides a method wherein the virus and/or the viralproteins comprise a protein that undergoes post-translational and/orperi-translational modification, such as glycosylation.

A good example of a viral vaccine that has been cumbersome to produce inany reliable manner is influenza vaccine. The invention provides amethod wherein the viral proteins comprise at least one of an influenzavirus neuraminidase and/or a hemagglutinin. Other viral proteins(subunits) and viruses (wt to be inactivated) or attenuated viruses thatmay be produced in the methods according to the invention includeenterovirus (such as rhinovirus, aphtovirus, or poliomyelitis virus),herpes virus (such as herpes simplex virus, pseudorabies virus or bovineherpes virus), orthomyxovirus (such as influenza virus), a paramyxovirus(such as Newcastle disease virus, respiratory syncitio virus, mumpsvirus or a measles virus), retrovirus (such as human immunodeficiencyvirus or a parvovirus or a papovavirus), rotavirus or a coronavirus(such as transmissible gastroenteritis virus), a flavivirus (such astick-borne encephalitis virus or yellow fever virus), a togavirus (suchas rubella virus or Eastern-, Western-, or Venezuelan equineencephalomyelitis virus), a hepatitis-causing virus (such as hepatitis Aor hepatitis B virus), a pestivirus (such as hog cholera virus), arhabdovirus (such as rabies virus), or a Bunyaviridae virus (such asHantavirus).

In one embodiment, a cell of the invention is useful in the generationof an influenza virus strain that does not grow very efficiently onembryonal eggs.

The invention also includes the use of a human cell having a sequenceencoding at least one adenoviral E1 protein or a functional derivative,homolog or fragment thereof in its genome, which cell does not producestructural adenoviral proteins for the production of a virus, or atleast one viral protein for use in a vaccine. Of course, for such a use,the cells preferred in the methods according to the invention are alsopreferred. The invention also provides the products resulting from themethods and uses according to the invention, especially viral proteinsand viruses obtainable according to those uses and/or methods,especially when brought in a pharmaceutical composition comprisingsuitable excipients and, in some formats, inactivated viruses, subunits,or adjuvants. Dosage and ways of administration can be sorted outthrough normal clinical testing in so far as they are not yet availablethrough the already registered vaccines.

Thus, the invention also provides a virus or a viral protein for use ina vaccine obtainable by a method or by a use according to the invention,the virus or the viral protein being free of any non-human mammalianproteinaceous material and a pharmaceutical formulation comprising sucha virus and/or viral protein.

The invention further provides a human cell having a sequence encodingat least one E1 protein of an adenovirus or a functional derivative,homolog or fragment thereof in its genome, which cell does not producestructural adenoviral proteins and having a nucleic acid encoding avirus or at least one non-adenoviral viral protein. This cell can beused in a method according to the invention.

In a preferred embodiment, the invention provides influenza virusobtainable by a method according to the invention or by a use accordingto the invention. In another embodiment, the invention providesinfluenza vaccines obtainable by a method according to the invention orby a use according to the invention.

In another aspect, the invention provides a kit for determining activityof a protease in a sample comprising at least one viral protein or virusobtainable by a method or a use of the invention, the virus or the viralprotein being free of any non-human mammalian proteinaceous material.This aspect of the invention is useful particularly for determiningprotease activity in culture medium. Culture medium is noted for being adifficult context for determining activity of a protease. However, byusing a viral protein or a virus of the invention as a target for theprotease, it is possible to accurately determine activity of theprotease also in culture medium. In a preferred embodiment, therefore,the protease activity is determined in a sample comprising culturemedium. In a preferred embodiment, the protease comprises trypsin. In apreferred embodiment, the viral protein comprises HA0.

In yet another aspect, the invention provides a method for concentratinginfluenza virus under conditions capable of, at least in part,preserving virus infectivity, comprising obtaining a cell-clearedsupernatant comprising the virus from a culture of cells, andultra-filtrating the supernatant under low shear conditions. Influenzavirus preparations harvested from embryonal eggs typically need to bepurified for the preparation of a vaccine. Purification typicallyentails at least one concentration step of the virus.

Current technologies for the concentration of influenza virus from suchrelatively crude preparations of influenza virus are cumbersome. Using amethod of concentration of the invention, it is possible to concentrateinfluenza virus preparations under conditions that maintain, at least inpart, infectivity of the virus. Preferably, virus is concentrated thatis or can be made infectious. “Can be made infectious,” as used herein,means the generation of infectious virus through cleavage of HA0.

In a preferred embodiment, the concentration is performed using a hollowfiber. A hollow fiber is particularly suited to concentrate under lowshear conditions.

In a preferred embodiment, the culture of cells comprises in vitrocultured cells. Particularly suited for concentration using a method ofthe invention is supernatant from in vitro cultured cells, particularlywhen the supernatant comprises serum-free culture medium. In a preferredembodiment, the ultra-filtration is performed with a filter allowingsingle proteins to pass while retaining the virus. Preferably, thefilter comprises a cut-off of 500 KD. More preferably, the filtercomprises a cut-off of 750 KD. In a particularly preferred embodiment,the concentration further comprises at least a partial removal ofproteins comprising a molecular weight smaller than 500 KD and, morepreferably, smaller than 750 KD. Preferably, the purification isachieved using a mentioned filter.

In yet another aspect, the invention provides infectious influenza virusor derivatives thereof concentrated with a method of the invention. Aderivative of an infectious influenza virus of the invention typicallyis a virus, virus particle, or viral protein or part thereof that can beused for immunization purposes. Typically, this entails a virusinfectivity inactivation step.

To further illustrate the invention, the following examples areprovided, which are not intended to limit the scope of the invention.

EXAMPLES Example 1

Materials and Methods

PER.C6 and MDCK Cell Culture

MDCK cells were cultured in Dulbecco's modified Eagle's medium (DMEM,Life Technologies Breda, NL) containing 10% heat-inactivated fetalbovine serum and 1×L-Glutamine (Gibco-BRL), at 37° C. and 10% CO₂.Suspension cultures of PER.C6 were cultured in ExCell 525 (JRHBiosciences) supplemented with 1×L-Glutamine, at 37° C. and 10% CO₂, instationary cultures in six-well dishes (Greiner) or in 490 cm² tissueculture roller bottles (Coming Costar Corp.) during continuous rotationat 1 rpm.

Immunofluorescence Test

Direct immunofluorescence assays for the detection of Influenza virusinfection were carried out using the IMAGEN™ Influenza Virus A and B kit(Dako) according to the standard protocol of the supplier. Samples wereviewed microscopically using epifluorescence illumination. Infectedcells were characterized by a bright apple-green fluorescence.

Propidium Iodide Staining

Cell pellets were resuspended in 300 μl of cold PBS/0.5% BSA+5 μl ofpropidium iodide (concentration 50 μg/ml) in PBS/FCS/azide solutionknown to persons skilled in the art. Viable and dead cells were thendetected via flow cytofluorometric analysis.

Hemagglutination Assay

In general, hemagglutination assays for Influenza virus titers wereperformed according to methods known to persons skilled in the art.Here, 50 μl of a two-fold diluted virus solution in PBS was added to 25μl PBS and 25 μl of a 1% suspension of turkey erythrocytes (BiotradingBenelux B.V.) in PBS and incubated in 96-well microtiter plates at 4° C.for one hour. The hemagglutination pattern was examined and scored andexpressed as hemagglutinating units (HAUs). The number of HAUscorresponded to the reciprocal value ofthe highest virus dilution thatshowed complete hemagglutination.

Western blot Analysis of the Influenza HA Protein

In general, obtained influenza viruses were disrupted in a Laemmlibuffer according to methods known in the art and different volumes ofobtained protein mixtures were separated using 10% SDS/PAGE gels. Inbrief, blots were blocked for 30 minutes at room temperature with blocksolution (5% nonfat dry milk powder (Biorad) in TBST supplemented with1% rabbit serum (Rockland), followed by three washes with TBST. Then,the blots were incubated with the anti-A/Sydney/5/97 HA antiserum(98/768 NIBSC) diluted 1/500 in 1%BSA/TBST with 5% rabbit serum(Rockland) O/N at room temperature. Again, the blots were washed eighttimes with TBST. Finally, the blots were incubated with the rabbitanti-sheep antiserum (HRP-labeled, Rockland) 1/6000 diluted in blocksolution for one hour at room temperature. After eight washes with TBST,the protein-conjugate complex was visualized with ECL (AmershamPharmacia Biotech), and films (Hyperfilm, Amersham Life Science) wereexposed. The antisera were obtained from the NIBSC (UK) and applied indilutions recommended by the NIBSC.

Single Radial Immunodiffusion (SRID) Assay

The concentration of hemagglutinin in supernatants, derived frominfluenza virus infected-PER.C6 cells, was determined by the singleradial immunodiffusion (SRID) test as previously described (Wood et al.1977). The assay was performed using standard NIBSC (UK) antigens andantisera reagents.

Plaque Assay

A total of 1 ml of ten-fold serially diluted viral supernatants wereinoculated on MDCK cells which were grown until 95% confluence insix-well plates. After one hour at 35° C., the cells were washed twicewith PBS and overloaded with 3 ml of agarose mix (1.2 ml 2.5% agarose,1.5 ml 2×MEM, 30 ml 200 mM L-Glutamine, 24 ml trypsin-EDTA, 250 ml PBS).The cells were then incubated in a humid, 10% CO₂ atmosphere at 35° C.for approximately three days and viral plaques were visually scored.

Virus Infectivity Assay (TCID₅₀)

Titration of infectious virus was performed on MDCK cells. In brief,cells were seeded in 96-well plates at a density of 4×10⁴ cells/well inDMEM supplemented with 2 mM L-Glutamine. Twenty-four hours later, cellswere infected with 100 μl of ten-fold serially diluted culturesupernatants, in quadruplicate, in medium containing Trypsin-EDTA at theconcentration of 4 mg/ml. Two hours after infection, cell monolayerswere washed two times in PBS and incubated in medium containing trypsinfor seven days at 35° C. Supernatants from these cultures were thentested in an HA assay. TCID₅₀ titers were calculated according to themethod of Karber (1931).

b-propiolactone Influenza Virus Inactivation

For inactivation of the viruses to obtain whole-inactivated virus forthe generation of vaccines derived from PER.C6, a mutation protocolknown to persons skilled in the art was performed using b-propiolactone.b-propiolactone is a very effective agent widely used for theinactivation of viruses and well known in the art for its mutatingeffects. It modifies nucleic acid bases of the viral genome and the hostcell genome and blocks replication thereafter. Following an establishedprotocol used to prepare the whole inactivated influenza vaccineprepared on embryonated eggs, the amount of virus corresponding toapproximately 400 mg of HA per strain was inactivated and used for thefinal vaccine formulation. Briefly, one volume of 0.3 M sodium phosphatebuffer was added to nine volumes of influenza virus preparation.Inactivation of the viruses was carried out adding one volume of 10% ofb-propiolactone (Newall Design, UK) to 100 volumes of phosphate-bufferedvirus preparation and incubated at 20° C. for 24 hours. Inactivation ofthe viruses was checked by plaque assay and no plaques were detected forany of the inactivated batches (data not shown).

Example 2A

PER.C6 Cell Banking and Pre-Culture

Cell line PER.C6, or derivatives thereof, were used. Cell lines werebanked by a two-tier cell bank system. The selected cell line was bankedin a research master cell bank (rMCB) which was stored in differentlocations. From this rMCB research, working cell banks (rWCB) wereprepared as follows: an ampoule of the rMCB was thawed and cells werepropagated until enough cells are present to freeze the cells by usingdry ice. Up to 500 ampoules containing 1 ml (1-2×10⁶ cells/ml) of rWCBwere stored in the vapor phase of a liquid N₂ freezer.

One ampoule containing 5×10⁶ PER.C6 cells of the WCB was thawed in awater bath at 37° C. Cells were rapidly transferred into a 50 ml tubeand resuspended by adding 9 ml ofthe suspension medium ExCell 525 (JRHBiosciences) supplemented with 1×L-Glutamine. After three minutes ofcentrifugation at 1000 rpm in a tabletop centrifuge, cells wereresuspended in a final concentration of 3×10⁵ cells/ml and cultured in aT80 tissue culture flask at 37° C. 10% CO₂. Two to three days later,cells were seeded into 490 cm² tissue culture roller bottles (ComingCostar Corp.), with a density of 3×10⁵ per ml and cultured in continuousrotation at 1 rpm.

Example 2B

PER.C6 Cells as Permissive Cell Line for Influenza A Virus

PER.C6 as a human cell was not known for its ability to sustaininfluenza virus infection and replication. It was, therefore, determinedwhether PER.C6 cells are permissive for influenza virus infection incomparison with the dog cell line MDCK that served as a positivecontrol.

On the day before infection, 2×10⁵ MDCK cells per well were seeded insix-well plates. Twenty-four hours later, 4×10⁵ seeded PER.C6 and theMDCK cells per well were infected with the H1N1 strain A/PuertoRico/8/34 (titer 3.6×10⁷ pfu/ml) (obtained from Dr. E. Claas, LeidenUniversity Medical Center, The Netherlands). Infection was performed atvarious multipliticies of infection (mois) ranging from of 0.1 to 10pfu/cell. After about two hours of incubation at 3 7° C., the inoculumwas removed and replaced by fresh culture medium. A directimmunofluorescence assay for the detection of influenza virus infectionwas performed 24 and 48 hours post-infection. The experiment showedpermissiveness of PER.C6 for influenza infection, with percentages ofpositive cells moi-dependent and comparable with MDCK (FIG. 1).

Example 3

PER.C6 Used for Influenza A Virus Propagation

It was verified whether replication and propagation of influenza viruscould be supported by PER.C6. On the day of infection, PER.C6 cells wereseeded in 490 cm² tissue culture roller bottles with the density of2×10⁵ cells/ml in a final volume of 40 ml in the presence of 5 μg/ml oftrypsin-EDTA (Gibco-BRL). Cells were either mock inoculated or infectedwith the H3N2 strain A/Shenzhen/227/95 (titer 1.5×10⁶ pfu/ml) (obtainedfrom Dr. E. Claas, Leiden University Medical Centre, The Netherlands).Infections were performed at moi 10⁻⁴ and 10⁻³ pfu/cell. After one hourof incubation at 37° C., the inoculum was removed by spinning down thecells at 1500 rpm and resuspending the cells in fresh culture medium +5μg/ml of trypsin-EDTA. Harvest of 1.3 ml of cell suspension was carriedout each day, from day 1 to day 6 post-infection. Supernatants werestored at −80° C. and used for hemagglutination assays. Cell pelletswere used for direct immunofluorescence tests and for propidium iodidestaining.

Example 4

Permissiveness of PER.C6 for Different Influenza Strains

To further investigate the permissiveness of PER.C6 for propagation ofvarious influenza strains, an infection byusing the HIN1 vaccine strainsA/Beijing/262/95 and its reassortant X-127, obtained from the NationalInstitute for Biological Standards and Control (NIBSC, UK), wasperformed. On the day of infection, PER.C6 cells were seeded in 490 cm²tissue culture roller bottles with the density of approximately 1×10⁶cells/ml in a final volume of 50 ml. Cells were inoculated with 5 μl(10⁻⁴ dilution) and 50 μl (10⁻³ dilution) of virus in the presence of 5mg/ml trypsin-EDTA. In order to establish if trypsin was indeedrequired, one more infection was carried out by inoculating 5 μl of thestrain A/Beijing/262/95 in the absence of the protease. Afterapproximately one hour of incubation at 37° C., the inoculum was removedby spinning down the cells at 1500 rpm and resuspending them in freshculture medium ±5 mg/ml of trypsin-EDTA. At day 2 and day 4post-infection, more trypsin was added to the samples. Harvest of 1.3 mlof cell suspension was carried out from day 1 to day 6 post-infection.Supernatants were stored at −80° C. and used for hemagglutination assaysand further infections; cell pellets were used for directimmunofluorescence tests. Results obtained with the above-mentionedimmunofluorescence and hemagglutination assays are shown in FIGS. 4 and5, respectively, illustrating the efficient replication and release ofthe viruses.

Example 5

Infectivity of Virus Propagated on PER.C6

It was verified whether the viruses grown in PER.C6 were infectious andif adaptation to the cell line could increase virus yields. Virussupernatants derived from PER.C6 infected with the strainsA/Beijing/262/95 and its reassortant X-127 (dil.10-3) and harvested atday6 post-infection were used. At the day of infection, PER.C6 wereseeded in 490 cm² tissue culture roller bottles, with the density ofapproximately 1×10⁶ cells/ml in a final volume of 50 ml. Cells wereinoculated with 100 μl and 1 ml of virus supernatant in the presence of5 mg/ml trypsin-EDTA. In order to establish if trypsin was stillrequired, one more infection was carried out by inoculating 100 μl ofthe strain A/Beijing/262/95 in the absence of the protease. Afterapproximately one hour of incubation at 37° C., the inoculum was removedby spinning down the cells at 1500 rpm and resuspending them in freshculture medium ±5 mg/ml of trypsin-EDTA. At day 2 and day 4post-infection, more trypsin was added to the samples. Harvest of 1.3 mlof cell suspension was carried out from day 1 to day 6 post-infection.Supernatants were stored at −80° C. and used for hemagglutination assaysand further infections; cell pellets were used for directimmunofluorescence tests. Results obtained with the above-mentionedimmunofluorescence and hemagglutination assays are shown in FIGS. 6 and7. Data obtained with the present experiment showed infectivity of theviruses grown in PER.C6 as well as an increase in virus yields.

Example 6

The Presence of Cell Surface Receptors for Influenza Virus on PER.C6

Propagation of human Influenza A and B strains in embryonated chickeneggs leads to a selection of receptor-binding variants that harbor aminoacid substitutions at the distal portion of the HA globular head in theexposed and functionally important regions ofthe molecule. Because ofthese mutations, the egg-adapted strains can differ from the originalhuman viruses in their antigenic and immunogenic activities, as well astheir virulence. Human influenza viruses isolated from MDCK cellsusually present an HA protein that is identical to the HA proteinpresent on the virus of the original clinical sample. A recent study(Govorkova 1999) clarified the molecular basis for the selection ofvariants in chicken eggs and the absence of this variant selectionphenomenon in MDCK cells. All human Influenza A and B strains isolatedfrom MDCK cells were found to bind with high affinity and specificityfor alpha2,6 sialic acid-galactose linkages present in oligosaccharidespresent in cell surface receptors, whereas their egg-grown counterpartsshowed an increased affinity for the alpha2,3 sialic acid-galactoselinkages in cell surface receptors carrying oligosaccharides(Sia2-3Gal). Using specific lectins, it was demonstrated that onlySia2-3Gal-containing receptors were present on the surface of chickenembryonic cells, whereas MDCK cells expressed both Sia2-6Gal andSia2-3Gal. The expression of the Sia2-3Gal and Sia2-6Gal moieties on thesurface of PER.C6 cells was studied by FACS analysis, using twodifferent digoxigenin-(DIG-) labeled lectins: Sambuca nigra agglutinin(SNA) that specifically recognizes Sia2-6Gal linkages and the Maackiaamurensis agglutinin (MAA), that specifically recognizes Sia2-3Gallinkages. FIG. 8A shows the recognition of the SNA and MAA lectins andtheir binding to the glycosylation sites. Furthermore, FIG. 8A shows theschematic interaction between the FITC-labeled anti-DIG antibody and theDIG-labeled lectin that recognizes the specific sialyl bond in theglycosylation backbone of the receptor present on the cell surface. Bothlectins were taken from the glycan differentiation kit (Boehringer-LaRoche).

The experiment was carried out on PER.C6 cells in suspension andadherent MDCK and CHO cells. MDCK and CHO cells were released from thesolid support using trypsin-EDTA (Gibco-BRL). The cell suspensions werethen washed once with Mem-5% FBS and incubated in this medium for onehour at 37° C. After washing with PBS (Gibco-BRL), the cells wereresuspended to a concentration of approximately 10⁶ cells/ml in bindingmedium (Tris-buffered saline, pH 7.5, 0.5% BSA, and 1 mM each of MgCl₂,MnCl₂ and CaCl₂). Cell aliquots were incubated for one hour at roomtemperature with the DIG-labeled lectins SNA and MAA. After one hour,lectin-treated cells were washed with PBS and incubated for anadditional hour at room temperature with FITC-labeled anti-DIG antibody(Boehringer-Mannheim). Finally, the cells were washed with PBS andanalyzed by fluorescence-activated cell sorting using a FAC-sortapparatus (Becton Dickinson). The results shown in FIG. 8B demonstratethat PER.C6 cells were stained by both lectins showing the presence ofthe Sia2-6Gal as well as the Sia2-3Gal receptors.

In the same experiment, MDCK cells were used as positive control forboth the sialylated receptors, whereas CHO cells, due to the absence ofthe alpha 2-6 sialyltransferase glycosylation enzyme in these hamstercells, represented a negative control for the Sia2-6Gal moiety. Theupper panels show results with the SNA lectin and the lower panelsshowing results with the MAA lectin. From these results, it can beconcluded that PER.C6 expresses cell surface proteins that have bothSia2-3Gal and Sia2-6Gal linkages in their oligosaccharide chains.

Example 7

Effect of Different Concentrations of Trypsin-EDTA on the Viability ofPER.C6 Cells, on the Influenza Virus Production in PER.C6 Cells and onthe Ha Protein Derived Thereof

Due to the absolute trypsin requirement for the propagation of influenzaviruses in cell cultures, the effects of different concentrations oftrypsin-EDTA on PER.C6 cell viability and virus replication in PER.C6cells after infection using several Influenza strains were investigated.

Infection with Influenza Virus Strain A/Sydney/5/97 in the Presence ofLow Concentrations of Trypsin

On the day of infection, PER.C6 cells were seeded in 490 cm² tissueculture roller bottles at a density of 1×10⁶ cells/ml in the presence oftrypsin-EDTA at final concentrations of 0.5, 1, 2, 3 and 5 mg/ml.

These trypsin concentrations did not interfere with the growthcharacteristics ofthe cells and their viability (data not shown). Cellswere either mock infected or infected with PER.C6-grown Influenza virusA/Sydney/5/97 at an moi of 10⁻⁴ pfu/cell. The viral production wasmonitored by direct immunofluorescence (data not shown),hemagglutination assays, single-radial-immunodiffusion (SRID) above andplaque assays, all as described above. Results from this experiment aredepicted in FIG. 9 and show that the HA content as measured by SRID, aswell as the biological activity of the virus expressed in HAU, werehighest when a trypsin concentration of 1 mg/ml was used. FIG. 9 alsoshows that by using a plaque assay the highest number of plaque formingunits (pfu) per ml was observed in the sample corresponding to cellsgrown in medium containing 2 mg/ml of trypsin.

Infection with Influenza Virus Strain B/Harbin/7/94

On the day of infection, PER.C6 cells were seeded in 490 cm² tissueculture roller bottles at a density of 1×10⁶ cells/ml in the presence ofdifferent concentrations of trypsin-EDTA ranging from 1 to 5 mg/ml.Cells were infected with PER.C6-grown virus B/Harbin/7/94 at an moi of10⁻³ pfu/cell. Production of the virus was monitored by directimmunofluorescence, hemagglutination and plaque assays as shown in FIG.10. The infectability of PER.C6 at day 2 increased with theconcentration of trypsin. At day 3, however, no significant differencewas observed in the percentage of infected cells when 1, 2.5 or 5 mg/mltrypsin was present. In the absence of trypsin (0 μg/ml), no influenzavirus infection was detected. At the day ofthe last harvest (day 4post-infection), the biological activity of the virus, as measured byhemagglutination assay, did not differ significantly. Interestingly, theinfectivity assay performed in samples that were taken at days 3 and 4after infection showed a difference in the production of the virus. Thehighest titers were obtained at day 3 and day 4 when a trypsinconcentration of 2.5 to 5 (day 3) and 1 mg/ml (day 4) were used.

Infection with Influenza Virus Reassortant X-127

On the day of infection, PER.C6 cells were seeded in T25 tissue cultureflasks at a density of 1×10⁶ cells/ml in the presence of differentconcentrations of trypsin-EDTA ranging from 0 to 7.5 mg/ml. Cells wereinfected with PER.C6-grown virus X-127 (egg-reassortant for the strainA/Beijing/262/95) at an moi of 10⁻⁴ and 10⁻³ pfu/cell. Viral growth wasmonitored by direct immunofluorescence, hemagglutination and plaqueassays. As shown in FIG. 11 and FIG. 12, HAU titers were identicalbetween samples, independent of the trypsin concentration and theinitial moi that was used. Furthermore, no significant differences wereobserved in the infectivity titers as measured by plaque assay.

Infection of PER.C6 with Influenza Virus Strain A/Sydney/5/97 in thePresence of High Concentrations of Trypsin

To test the effect of increasing concentrations of trypsin on viabilityof the cells and virus replication, PER.C6 cells were seeded in rollerbottles at a density of 1×10⁶ cells/ml in the presence of variousconcentrations of trypsin-EDTA ranging from 0 to 12.5 μg/ml. Cells wereeither mock infected or infected with PER.C6-grown virus A/Sydney/5/97virus at an moi of 4×10⁻⁵ pfu/cell. HAU's presence in the obtainedbatches were determined as described. Importantly, data depicted in FIG.13 clearly show that trypsin concentrations up to 10 μg/ml do notinterfere with the cell viability. Moreover, the biological activity ofthe virus obtained at day 4 after infection as measured by HAU washigher when a trypsin concentration of 2.5 to 5 μg/ml was used.Furthermore, the TCID₅₀ was measured (FIG. 14, graph portion A) andplaque assays were performed (data not shown). No relevant differenceswere found in these plaque assays, in the infectivity titers (TCID₅₀),in the HA cleavage and quantity (approximately 10 μg/ml) as determinedby western blot analysis shown in FIG. 14B.

Example 8

Influenza Virus Production on PER.C6 Cells in a Hollow Fiber-PerfusionBioreactor System

The scalability of influenza virus production in suspension growingPER.C6 cells was studied by using 3-liter (total volume) bioreactorscontaining a 2 liter cell suspension volume in serum-free medium, whichis also free of animal- or human-derived proteins (ExCell 525, JRHBiosciences).

Influenza infection was carried out at a cell density of approximately3×10⁶ cells/ml. Cells were inoculated with PER.C6-grown A/Sydney/5/97virus, at an moi of 10⁻⁴ pfu/cell. Samples of 5 to 10 ml of cellsuspensions were taken every day to perform general cell counts, todetermine the viability of the cells, for glucose concentrationmeasurements, for direct immunofluorescence, for hemagglutination andfor infectivity assays. The results of these experiments are shown inFIG. 15.

To investigate the presence and the status of the HA protein westernblots using two different anti-HA antibodies obtained from NIBSC wereused. SRID assays as described above were also performed. The resultsdepicted in the two western blots in FIG. 16 show that the Influenzavirus batch produced in this bioreactor yielded an HA content of anestimated concentration of 15 μg/ml which was confirmed by SRID assays.The HA produced is comparable to reference NIBSC HA in terms of subunitcomposition and immune reactivity with the reference subtype-specificantisera.

Example 9

Infection of PER.C6 with A/Sydney/5/97 in a 15 Liter Bioreactor Followedby a specific Down Stream Process (DSP)

Suspension growing PER.C6 cells was incubated at 37° C. in a 15-literbioreactor hollow fiber perfusion system, with a cell suspension volumeof 10 liters in serum-free ExCell 525 medium (JRH Biosciences).Influenza infection was carried out at 35° C. at a cellular density ofapproximately 3.3×10⁶ cells/ml in medium containing 5 mg/ml trypsin-EDTA(Life Technologies). Cells were inoculated with PER.C6-grownA/Sydney/5/97 virus (passage number 3) at an moi of 10⁻⁴ pfu/cell.Perfusion with serum-free ExCell 525 medium containing trypsin-EDTA wascontinued during the first 24 hours upon infection. Two dayspost-infection, cells were fed with a fed-batch solution containingglucose, essential amino acids and extra glutamine: 82 ml per litersuspension containing 50 m/v % glucose (NPBI-The Netherlands), 50×essential ammino acids without Gln (Gibco-BRL-Life Technologies) and 200mM glutamine (Gibco-BRL-Life Technologies). Cell suspension samples of10 ml were taken every day in order to perform standard cell counts(results shown in FIG. 17, left graph), glucose concentrationmeasurements (results shown in FIG. 17, right graph), directimmunofluorescence (FIG. 18), hemagglutination (FIG. 19) and infectivityassays (data not shown). Furthermore, the HA protein was investigated bywestern blot analysis and compared to an NIBSC standard HA control (FIG.20). On the day of the final harvest of the entire cell suspension (92hours post-infection), a cell debris clarification was performed in acontinuous flow at 20,000 g using the Powerfuge™ separation system(Carr, JM Separations) according to the protocols provided by themanufacturer. Clarified supernatant was then concentrated twenty-foldusing a hollow fiber membrane cartridge of 500 kD cut-off (A/GTechnology, JM Separations). The results depicted in FIG. 21 show thatthe quantitative recovery of live influenza virus after concentration byhollow fiber as measured by hemagglutination and infectivity assays isvery significant.

Example 10

The Immunogenicity of PER.C6-Grown Influenza Viruses and VaccinesDerived Therefrom

To determine the immunogenicity of PER.C6-grown influenza viruses, an invivo study and challenging model in ferrets was designed. Two batches oftrivalent whole-inactivated influenza vaccine (composed ofA/Sydney/5/97, A/Beijing/262/95 and B/Harbin/7/94), containing 15 μg HAof each of the three strains, were used. The first batch was obtainedfrom fertile hens' eggs and the second was obtained from PER.C6 cells.Production, purification, inactivation and formulation of the trivalentwhole-inactivated PER.C6-derived Influenza vaccines were performed asdescribed below.

Growth of A/Sydney/5/97, A/Beijing/262/95 and B/Harbin/7/94 Influenzastrains on PER.C6

Production of all three influenza viral batches were performed in threeseparate 3-liter hollow fiber fed-batch bioreactor systems with cellsuspension volumes of 2 liters. Fed-batch was performed with theaddition of the following solution: A total volume of 96 ml containing50 m/v % glucose (NPBI), 50× essential amino acids without Gln(Gibco-BRL-Life Technologies), 200 mM glutamine (Gibco-BRL-LifeTechnologies) and 7.5 m/v % NaHCO₃ (Merck) was added once. Influenzainfections were carried out at cell densities ranging from 1.8×10⁶ to2.6×10⁶ viable cells/ml, in ExCell 525 serum-free medium containing 5mg/ml trypsin-EDTA. PER.C6 cells were inoculated with the PER.C6-grownA/Sydney/5/97, A/Beijing/262/95 and B/Harbin/7/94 virus batches atdifferent mois: 10⁻⁴ (A/Sydney/5/97) or 10⁻³ (A/Beijing/262/95 andB/Harbin/7/94) pfu/cell. During the virus production period, samples of10 ml were taken every day to perform standard cell and viabilitycounts, glucose concentration measurements, direct immunofluorescenceand Hemagglutination assays. FIG. 22 (results from theA/Sydney/5/97-infected PER.C6 cells) shows the total and viability cellcounts after infection with the virus (upper left panel), the glucoseconsumption (upper right panel), the percentage of positive cells in thedirect immunofluorescence detection (lower left panel) and the HAUs(lower right panel). FIG. 23 (results from the A/Beijing/262/95-infectedPER.C6 cells) shows the total and viability cell counts after infectionwith the virus (upper left panel), the glucose consumption (upper rightpanel), the percentage of positive cells in the directimmunofluorescence detection (lower left panel) and the HAUs (lowerright panel). FIG. 24 (results from the B/Harbin/7/94-infected PER.C6cells) shows the total and viability cell counts after infection withthe virus (upper left panel), the glucose consumption (upper rightpanel), the percentage of positive cells in the directimmunofluorescence detection (lower left panel) and the HAUs (lowerright panel). Virus-containing concentrates were stored at −80° C. untilDSP.

In all three cases, the glucose consumption, viability and total cellcounts of the PER.C6 cells were comparable. Also, the production levelsof the three viruses, as measured by direct immunofluorescence, weresimilar. Although the HAU and infectivity titers differed betweendifferent strains, PER.C6 sustained replication of all influenza virusesthat were tested here.

On the day of harvest ofthe entire batch (either at day 3 or at day 4post-infection), viral supernatants were clarified by centrifugation at2000 rpm in a table top centrifuge and concentrated ten-fold byultra-filtration using a hollow fiber membrane cartridge of 750 kDcut-off (A/G Technology, JM Separations) following the protocolsprovided by the manufacturer. Influenza viruses were purified from theconcentrated supernatants via two subsequent density centrifugationsteps: a 25-70% block sucrose gradient (1.5 hours at 27K) followed by acontinuous 25-70% sucrose gradient (four hours at 23K). Viral bands werediluted in approximately 50 ml of a Phosphate buffer and finallypelleted at 24,000 rpm in an ultracentrifuge. Viral pellets weredissolved in 1.5 to 2.3 ml of a Phosphate buffer, aliquoted and frozenat −80° C.

The formulation of inactivated Influenza vaccines is based on the amount(in micrograms) of the “immunologically active” HA protein, as measuredby the SRID assay (Wood et al. 1977). The test was performed tocharacterize the HA content of the batches. At the same time, totalamount of proteins was measured using the Lowry-based DC-protein assaykit (Biorad) following the procedures suggested by the manufacturer. Itwas found that HA constitutes about 20 to 30% of the total proteincontent of the virus preparation.

Example 11

In vivo Immunogenicity of Inactivated Vaccines Produced in Eggs and onPER.C6

Ferrets and mice represent two well-established animal models forstudying influenza infection and have been used to determine theefficacy and immunogenicity of influenza vaccines. Using the mouse modeltest system, the immunogenicity produced by the PER.C6 and egg-derivedtrivalent vaccines containing A/Sydney/5/97, A/Beijing/262/95 andB/Harbin/7/94 are compared by analyzing sera of vaccinated animals byHemagglutination inhibition assay. Using the ferret infection model,immunization is followed by a challenge with A/Sydney/5/97. Virusrecovery on MDCK cells and Hemagglutination inhibition assay performedon the sera are used to compare the immunogenicity and efficacy of thetwo vaccines.

In Vivo Study in Mice

Ninety female Balb/C mice are divided into nine groups of ten mice. Onday 0, up to 100 ml of blood is collected. The serum is separated andstored at −20° C. Each mouse is then vaccinated with the appropriatevaccine according to the schedule in Table I. On day 28, a further 100ml of blood is taken. Serum is stored at −20° C. Each mouse is againvaccinated according to the schedule in Table I. On day 42, a 100 mlblood sample is taken and all mice are sacrificed. Serum is separatedand frozen at −20° C. Hemagglutination Inhibition (HI) assays areconducted on serum samples from day 0, 28 and 42. All these assays areconducted in parallel for each day for both egg- and cell-grown viruses.TABLE I Immunogenicity test in mice. IMMUNI- GROUP ZATION VACCI- TOTALNUMB- ANTIGEN VOLUME NATION mg HA ER TYPE (ml) ROUTE per dose 1 Eggtrivalent whole virion 0.5 s.c. 9.0 2 Egg trivalent whole virion 0.5s.c. 3.0 3 Egg trivalent whole virion 0.5 s.c. 1.5 4 Egg trivalent wholevirion 0.5 s.c. 0.15 5 PER.C6 trivalent whole virion 0.5 s.c. 9.0 6PER.C6 trivalent whole virion 0.5 s.c. 3.0 7 PER.C6 trivalent wholevirion 0.5 s.c. 1.5 8 PER.C6 trivalent whole virion 0.5 s.c. 0.15 9 PBS0.5 s.c. 0In Vivo Study in Ferrets

Eighteen adult female ferrets (albino or polecat) were divided in threegroups of six divided as follows: Group 1 received the egg-derived testvaccine intramuscularly (IM), the animals were challenged withA/Sydney/5/97. Group 2 received the PER.C6-derived test vaccine IM, theanimals were challenged with A/Sydney/5/97. Group 3 received the testvaccine diluent only and were challenged with A/Sydney/5/97. On days 0and 28, the test vaccines were administered. On day 56, all the ferretswere infected intranasally with 0.5 ml of the A/Sydney/5/97 challengevirus at TCID₅₀ 10³. Nasal washes were performed and inflammatory cellcounts, temperature and weights of the ferrets were monitored once dailyfrom day 57 to 63. All animals were sacrificed on day 63. Serum wasseparated and stored at −20° C. The nasal wash samples were stored onice and a nasal wash recovery cell count was performed using Trypan blueexclusion assay.

The titer of the virus obtained from the nasal wash samples wasdetermined by measuring the virus recovery on MDCK cells. The Spearmanand Karber (1931) calculation was used to calculate TCID₅₀ values.Hemagglutination inhibition analyses were conducted on serum samplestaken on day 0, 28, 56 and 63. From this experiment, it was concludedthat the PER.C6-derived test vaccine was effective.

Example 12

Characterization of HA Protein Derived from Influenza Virus Produced onPER.C6

In order to study the glycosylation of HA in PER.C6 cells, a batch ofuncleaved HA (HA0) was generated. PER.C6 cells were infected with virusA/Sydney/5/97 (passage number 5 on PER.C6) at mois of 1, 0.1 and 0.01pfu/cell in ExCell 525 medium containing trypsin-EDTA at the finalconcentration of 5 mg/ml. After one hour of incubation at 35° C., cellswere washed twice with PBS to remove trypsin and incubated O/N at 35° C.and 10% CO₂, in the absence of trypsin. The next day, cell suspensionswere harvested and centrifuged (500 g) and cell pellets were washedtwice with medium. Viral supernatants were frozen at −80° C. and samplesthereof were used in western blot assays as described to investigate thepresence or absence of uncleaved HA protein. Uncleaved HA protein (HA0)consists of the two subunits: HA1 and HA2, that are connected via adisulfide bond. Since this disulfide bond can be disrupted by reductionwith DTT, HA1 and HA2 can be separated and visualized on a reducing gelfollowed by western blots using antisera that recognize the subunits. Ifthe HA protein consists only of HA0, one band will be visible thatmigrates slower through an SDS/PAGE gel as compared to the HA1 subunitand the fastest migrating HA2 subunit. The results shown in FIG. 25suggest the presence of mainly uncleaved HA0 from PER.C6 infections whencompared to the egg-derived positive control that was obtained from theNIBSC (UK). To confirm that the band detected was indeed uncleavedhemagglutinin, an HA0 sample was digested with different concentrationsof trypsin ranging from 2.5 to 10 μg/ml in medium O/N at 37° C. Thedigested proteins were then loaded under reducing conditions on anSDS/PAGE gel followed by western blot analysis using the same antiseraas described for FIG. 14. As shown in FIG. 26A, cleavage of the HA0could be achieved, confirming the generation of uncleaved HA protein onPER.C6. Based on these results, an assay to determine trypsin activityin culture medium, using Influenza HA0 as substrate is developed.

Trypsin Activity Assay

To determine whether trypsin, present in the culture medium of anInfluenza production run is still active, a trypsin activity assay hasbeen developed. This assay is based on the measurement of the enzymaticactivity of trypsin to cleave the substrate that is most relevant forinfluenza vaccine production: the HA0.

It was determined whether, in a culture of PER.C6 inoculated withInfluenza B/Harbin/7/94 (moi 10⁻³/10⁻⁴ pfu/cell), the trypsin remainedactive over the entire production run. To this end, 10 μl of supernatanttaken at day 1, 2 and 3 post-infection were used to cleave 68 ng of thesubstrate that consisted of HA0 of Influenza A Sydney/5/97 virus, O/N at37° C. Following digestion, protease inhibitors were added to a finalconcentration of 1× (complete protease inhibitor cocktail, BoehringerMannheim) in 3× Laemli buffer with 150 mM DTT (Fluka). The samples wereloaded on a 10% Tris-HCL SDS/PAGE gel (Biorad) and run. The western blotwas performed as described. The results are shown in FIG. 26B, anddemonstrate that in cultures of PER.C6 inoculated with InfluenzaB/Harbin virus, trypsin remained active during the entire production runas culture supernatants were able to cleave HA0 completely.

Example 13

Digestion of HA0 with N-Glycosidase F

The influenza virus HA protein is a glycoprotein that contains three tonine N-linked glycosylation oligosaccharide sites. The number of sitesdepends on the virus strain. The location of these sites is determinedby the nucleotide sequence of the HA gene and since the viral genome ofInfluenza is replicated by an error-prone RNA polymerase, mutations thatgenerate the addition or removal of glycosylation sites occur at highfrequency. The composition and structure of the oligosaccharide chainspresent on the HA is then determined by the array of biosynthetic andtrimming glycosylation enzymes provided by the host cell. Sinceglycosylation of HA plays an important role in virulence and vaccineefficacy, the properties of HA produced on Influenza infected PER.C6 wasinvestigated. A digestion of A/Sydney/5/97 uncleaved HA0 protein withthe N-glycosydase F enzyme was performed using protocols provided by themanufacturer to remove the seven oligosaccharides expected to be presenton the A/Sydney/5/97 HA polypeptide. Influenza A/Sydney/5/97 was lysedwith 1% Triton X-100 (Merck). Protease inhibitor was added to an aliquotof this lysed virus corresponding to 68 ng of HA, to a finalconcentration of 1× (Complete Protease Inhibitor Cocktail BoehringerMannheim). This sample was incubated in the presence of 100 mM NaPO₄ pH7, 10 mM EDTA (J. T. Baker), 1% SDS (J. T. Baker) and 1%B-mercaptoethanol (Merck). This was incubated for ten minutes at roomtemperature. The sample was diluted five times in mM NaPO₄ pH 7, 10 mMEDTA (J. T. Baker), 0.625% NP-40 and 1% B-mercaptoethanol (Merck). Ofthis, 40 μl was used for the glyco-F digestion. For this, 2 μl 1 U/μl ofglyco-F (N-Glycosidase F, Boehringer) was added and incubated for aminimum period of 16 hours at 37° C. Then 3× Laemli buffer with 150 mMDTT (Fluka) was added to a final concentration of 1×. The samples wererun on a 7.5% SDS/PAGE gel. The SDS-Page and western blot were performedas follows. In brief, the blot was blocked for 30 minutes at roomtemperature with block solution (5% nonfat dry milk powder, Biorad inTBST supplemented with 1% rabbit serum (Rockland) followed by threewashes with TBST. Then, the blot was incubated with the anti-A/Sydney/5/97 HA antiserum (98/768 NIBSC) diluted 1/500 in 1% BSA/TBSTwith 5% rabbit serum (Rockland) overnight at room temperature. Again,the blot was washed eight times with TBST. Finally, the blot wasincubated with the rabbit anti-sheep antiserum-HRP-labeled (Rockland)1/6000 diluted in block solution for one hour at room temperature. Aftereight washes with TBST, the protein-conjugate complex was visualizedwith ECL (Amersham Pharmacia Biotech) and films (Hyperfilm, AmershamLife Science) were exposed. As shown in FIG. 27, treatment with theglycosidase-F enzyme clearly reduced the size of the protein withapproximately 28-30 kD, being approximately the predicted loss of about4 kD per oligosaccharide. The protein band depicted with an asterisk (*)is the de-glycosylated HA0 that migrates similarly to the HA1 subunitproduct obtained after cleavage of HA0 into HA1 and HA2 subunits (rightlanes).

Example 14

Cleavage of HA0 with Accutase

The possibility of replacing the mammalian-derived trypsin-EDTA withnon-mammalian or recombinant proteins was investigated. Recently, amixture of proteolytic and collagenolytic enzymes (Accutase™, PAA) frominvertebrate species became available for routine cell culture. Due toits non-mammalian source, Accutase is free of prions, parvovirus, andother components that potentially can contaminate trypsin-EDTAsolutions. No information regarding the type of proteases present inAccutase could be obtained to date. The cleavage of HA0 was studiedusing western blot analysis. A constant amount of HA0 protein, obtainedby PER.C6 infected with A/Sydney/5/97 at an moi 1 pfu per cell withouttrypsin, was digested with serial dilutions of Accutase, O/N at 37° C.As a positive control, the same amount of HA0 digested with 100 ng oftrypsin-EDTA was used. The digested proteins were then loaded on a 10%SDS-PAGE gel, under reducing conditions, for western blot analysis. Asshown in FIG. 28, digestion with 2 ml of Accutase treatment resulted incomplete cleavage of HA0; partial cleavage was observed using 0.2 ml.These results suggest that treatment with Accutase during influenzareplication and production can replace trypsin-EDTA during influenzainfections on PER.C6.

Example 15

Electron Microscopy Analysis of Influenza Viruses on PER.C6 Cells

Transmission electron microscopy studies were done on PER.C6 cells thatwere infected with the influenza strain A/Sydney/5/97, as well as onviral-containing supernatants and sucrose purified material, todetermine the phenotype of this influenza virus produced on PER.C6. Allmethods that were used are well known to persons skilled in the art.FIG. 29 shows that the last stages of the virus life cycle arerepresented by budding and release of enveloped virions from thecytoplasmic membrane. Spikes corresponding to the HA and NA viralproteins were detected, ornamenting the periphery of the virionparticles. The figure also shows the characteristic pleiomorphism ofinfluenza viruses.

Example 16

Infection of PER.C6 with a Large Variety of Influenza A and B VirusStrains

The use of PER.C6 as a platform technology for the production ofinfluenza vaccine would preferably require PER.C6 to support the growthof a wide range of strains of different influenza subtypes.

Static suspension cultures of PER.C6 cells that were grown in T25 flasksand/or in six-well plates in ExCell 525 medium, were infected at a celldensity of 10⁶ cells/ml with 16 different strains of influenza viruses(FIG. 30A). These strains comprised several H3N2, H1N1, B type and Avianstrains. Infections were performed in the presence of 5 μg/ml oftrypsin. The viruses were obtained from NIBSC as egg-passaged wt orreassortant strains and are noted. Infection was performed with a virusdilution recommended by the NIBSC in the product sheets that weredelivered with the different strains. All viruses tested were capable ofpropagation on PER.C6 as visualized by immunofluorescence (data notshown) and titration of supernatant fluids in pfu assay (FIG. 30B).

These results show that even influenza strains (depicted by anasterisk), such as A/Johannesburg/33/94, B/Beijing/184/93 andA/Duck/Singapore-Q/F119-3/97, which are normally very difficult toproduce on embryonated eggs, can replicate and be produced on PER.C6cells.

Example 17

Generation of Herpes Simplex Type 1 (HSV-1) Virus, Herpes Simplex type 2(HSV-2) Virus and Measles Virus on PER.C6

It was tested whether viruses other than influenza virus and adenovirus,for example, Herpes Simplex Virus type 1 and 2 and measles virus, couldalso replicate on PER.C6. Vaccines that are derived from thesePER.C6-grown viruses and that induce neutralizing effects in humans forprotection against wt infections, are generated from the PER.C6-grownvirus batches. The strains that were obtained from ATCC and used forinfection of PER.C6 cells are depicted in Table II. TABLE II Herpessimplex virus and Measles strains that were obtained from the ATCC andthat were used for infection of PER.C6 cells. ATCC Passage Virus Straincat no. Lot no. history Titer Herpes Macintyre VR-539 1327850 y.s./12,10^(6.75) Simplex PR TCID50/200 μl Type 1 RabK/5, Mb/1, PrRabK/5,Vero/4, Vero (ATCC CCl-81)/1 Herpes MS VR-540 216463 Sheep 10^(7.5)Simplex choroid TCID50/200 μl Type 2 plexus/?, HeLa/?, PrRabK/7, Vero(ATCC CCl-81)/3 Measles Edmonston VR-24 215236 HK/24, 10⁴ HuAm/40,TCID50/ml MRC-5/1, MRC-5 (ATCC CCL- 171)/1

To test whether HSV-1, HSV-2 and measles viruses obtained from the ATCCcould replicate and be produced on PER.C6, passage number 46 cells wereseeded in Labtek chambers, coated with Poly-L-Lysine using knownmethods, at 10⁵ cells/well. Monkey-derived Vero cells (obtained fromATCC) were cultured at passage number 137 and were used as positivecontrols and seeded at a density of 2.5×10⁴ cells/well. At day 0, whenwells with PER.C6 cells were 60% and Vero cells 80% confluent, cellswere infected with different mois (10⁻³, 10⁻², 10⁻¹ and 1 TCID₅₀ percell). At daily intervals upon infection, cells were fixed and assayedin immunofluorescence using FITC-conjugated type-specific monoclonalantibodies using a kit (Imagen Herpes Simplex Virus (HSV) Type 1 and 2,Dako- and FITC-conjugated antibodies against the HA and matrix proteinof measles virus (measles IFA kit, Light diagnostics), following theprocedures suggested by the manufacturer. The antisera are directedagainst HSV-1 and −2 and Measles virus antigens.

The results summarized in FIG. 31 show that PER.C6 is permissive forHSV-1 (FIG. 31, portion D), HSV-2 (FIG. 31, portion E) and measles virus(FIG. 31, portion A) infections. Furthermore, the kinetics suggest thatthese viruses replicate on PER.C6 in an moi-dependent manner.

Next, it was investigated whether HSV-1, -2 and measles virus could bepropagated on PER.C6. To this end, cells were infected with moi of 0.01,0.1 and 1 TCID₅₀/cell for HSV-1 (FIG. 32 lower portion) and HSV-2 (FIG.32 upper portion) and an moi of 0.001 TCID₅₀/cell for measles virus(FIG. 32 middle portion) (passage number 1). At the occurrence of almostcomplete CPE, cells and supernatants were harvested, quickly frozen inliquid N₂, and thawed. After this, clarified supernatants were passagedblindly using approximately 100 μl to PER.C6 (this is passage number 2).After reaching almost complete CPE again, a third passage (passagenumber 3) was performed in a similar manner. The mois of the passagenumber 2 and 3 were determined in retrospect by TCID₅₀ assays.

The results of these experiments show that Herpes Simplex Virus type 1and −2 and Measles viruses can be replicated on PER.C6 and thatreplication and propagation can even occur when mois as low as 10⁻⁷ areused.

Example 18

Screening of Rotavirus for Replication on PER.C6

To test whether PER.C6 could also support the replication of arotavirus, PER.C6 cells were infected with a rhesus rotavirus (MMU18006; ATCC#VR-954; strain S:USA:79:2; lot # 2181153). PER.C6 cells(passage number 41) were cultured at a density of 1×10⁵ per ml andmonkey-derived Vero cells (obtained from ATCC, passage number 139) werecultured at a density of 2.5×10⁴ per ml and subsequently seeded inLabtek chambers that had been pre-coated with poly-L-Lysine aspreviously identified. Cells were infected with an moi of 1 TCID₅₀/cellof Rhesus rotavirus in the presence and absence of 2 μg/ml oftrypsin-EDTA. After 90 minutes of infection, cells were washed withExCell 525 medium and further incubated at 37° C. at 10% CO₂ in ahumidified atmosphere. On five consecutive days following infection,samples of supernatants were harvested, clarified from cells and celldebris by centrifugation at 2000 rpm in a table top centrifuge andanalyzed in an ELISA specific for rotavirus (IDEIA Rotavirus, Dako). Theresults depicted in FIG. 33 clearly show that Rhesus rotavirusreplicates on PER.C6.

Example 19

Growth of Pandemic Influenza Strains on E1 Transformed Cells

In FIG. 30A, it was outlined which influenza strains could be grown onmammalian El-transformed cells, such as PER.C6® cells. Clearly, the H3N2and H1N1 strains as well as the influenza B strains and the avianstrains could all be grown to significant titers on PER.C6 cells.

It is known that only a limited number of influenza strains generallycause disease in humans. However, it is now well established that alsostrains that generally only occur in birds can, through re-assortment inman, become infectious for humans and be transferred from one humanbeing to another. Such bird-derived influenza strains can causeso-called “pandemics” and infect millions of people around the world andcause countless numbers of casualties. One major example of suchpandemic was the Spanish flu that killed millions around 1918. In TableIII (partly taken from: “Avian influenza: assessing the pandemic threat”publication from the World Health Organization (January 2005)), previousworldwide outbreaks of highly pathogenic avian influenza strains aregiven. In Table IV (from the same source), documented human infectionswith avian influenza viruses are listed. From these tables, it is atleast clear that at least the following virus strains are a threat tothe well being of birds and humans worldwide: H5N1, H5N2, H5N8, H5N9,H7N1, H7N3, H7N4, H7N7, and H9N2. On top of this, it has recently beenestablished through reverse genetics that the Spanish flu in 1918-1919was caused by the H1N1 variant (Tumpey T M et al. (2005) Science310:77-80).

To date, no vaccines exist against the pandemic strains listed in TableIII and IV. It is therefore highly preferred to generate vaccinesagainst influenza strains that are not only involved in the yearlyinfluenza outbreaks for which numerous vaccines from different vaccineproducers are available, albeit only produced on embryonated hens' eggs,but also against the strains that are associated with pandemic outbreaksor that have the potential to be associated with a pandemic outbreak (aslisted above). Generally, these strains have a H5, H7 or a H9haemagglutinin. Clearly, it cannot be excluded that new combinations ofhaemagglutinin and neuraminidase will arise and mutate to become highlypathogenic in addition to the combinations listed above.

Here, it was tested whether a number of these strains could be grown onE1-transformed mammalian cells, such as the human retinoblast-derivedPER.C6 cells. The strains that were used as examples were H5N1, H7N7 andH9N2.

For this, H5N1 (A/Hong Kong/97) was initially grown on MDCK cells andsubsequently inoculated in PER.C6 cell cultures. H7N7(A/Chicken/Netherlands/03) and H9N2 (A/Chicken/Saudi Arabia/00) strainswere initially grown on embryonated eggs and subsequently inoculated inPER.C6 cell cultures, generally as described herein. Fluorescence wasdetermined as outlined above. FIG. 34 indicates that H5N1 and H9N2infected almost all cells as nearly 100% of the cells expressed thevirus proteins. H7N7 was only determined in approximately 10% of thecells. Since the titers of the viruses were not known, it might be thatthe titer of the H7N7 batch was too low to obtain a proper result.

Subsequent experiments on PER.C6 cells were generally performed asdescribed above, in which the inoculation titers were m.o.i. 0.0001(H5N1 and H7N7) and 0.0006 (H9N2). Infection rates were determined byTCID50. This experiment was performed twice. The TCID50 was determinedusing methods known to the person skilled in the art, on MDCK cells, asdescribed above. The results are given in FIG. 35. Clearly, theE1-transformed PER.C6 cells are able to sustain the growth of all threestrains that are associated with a pandemic outbreak, indicating thatPER.C6 cells are a suitable substrate to produce influenza viruses basedon these strains and to make vaccines against pandemic influenzastrains.

In a next experiment, different m.o.i.'s were applied using the H5N1strain. PER.C6 cells were inoculated with m.o.i. 1, 0.1, 0.01, 0.001,0.0001 and 0.00001 (given as 1, −1, −2, −3, −4 and −5). TCID50 valueswere again determined with MDCK cells. The results are shown in FIG. 36and clearly indicate that even at very low inoculation titers goodresults can be achieved, as an m.o.i. of 0.01 gave the best resultsafter three days upon inoculation. These results clearly show thatPER.C6 cells are very suitable for the growth of pandemic influenzastrains in tissue culture thereby circumventing the need for vaccineproduction using embryonated hen's eggs.

The invention therefore also relates to vaccines comprising influenzaviruses that are replicated and grown on E1-transformed cells,exemplified by PER.C6 cells. Influenza vaccines may be produced byattenuating produced viruses, inactivating produced viruses ordisintegrating produced viruses, wherein it is preferred that theviruses are split and the two major immunogenic components of the virus,haemagglutinin and neuraminidase are further purified and finally mixedwith a therapeutically acceptable excipient, such as a therapeuticallyacceptable buffer or salt solution. Such therapeutically acceptableexcipients or carriers are well known in the art. Vaccines may compriseadjuvants, such as aluminum-based adjuvants (aluminum hydroxide and/oraluminum phosphate). Preferably, the split influenza virus containingvaccines, according to the invention, also comprise an adjuvant tostimulate the immune response. The viruses obtained by the methodsapplying adenovirus E1-transformed cells are typically harvested after 2to 10 days after inoculation and after harvest attenuated, inactivatedor split by methods known to the person skilled in the art. Harvest maybe accompanied by sonication to release more virus. Also, contaminatingnucleic acids are typically removed by a nuclease treatment, such asBenzonase. Formalin or BPL may be used for inactivation. Triton ispreferably used for obtaining a split virus. The split-virus basedvaccines typically comprise the antigenic determinants based on the HAand the NA components of the viral coat, which components are typicallypurified by one or more chromatography steps, including anion and/orcation exchange resins and several concentration, zonal centrifugationand dia- and/or ultra-filtration steps. The formulation of the vaccineis such that the components are therapeutically acceptable in mammals,and preferably in humans.

Although the invention has been described with a particular amount ofdetail and with respect to particular examples, the scope of theinvention is to be determined by the appended claims. TABLE IIIOutbreaks of highly pathogenic avian influenza virus strains around theworld. period Location strain 1959 Scotland, United Kingdom H5N1 1963England, United Kingdom H7N3 1966 Ontario, Canada H5N9 1976 Victoria,Australia H7N7 1979 Germany H7N7 1979 England, United Kingdom H7N71983-1985 Pennsylvania, USA H5N2 1983 Ireland H5N8 1985 Victoria,Australia H7N7 1991 England, United Kingdom H5N1 1992 Victoria,Australia H7N3 1994 Queensland, Australia H7N3 1994-1995 Mexico H5N21994 Pakistan H7N3 1997 Hong Kong H5N1 1997 New South Wales, AustraliaH7N4 1997 Italy H5N2 1999-2000 Italy H7N1 2002 Hong Kong H5N1 2002 ChileH7N3 2003 Netherlands H7N7 2004 Pakistan H7N3 2004 Texas, USA H5N2 2004British Colombia, Canada H7N3 2004 South Africa H5N2 2003-2005 SouthEast Asia H5N1 2005 Turkey H5N1 2005 Romania H5N1

TABLE IV Documented human infections with avian influenza viruses.period location strain cases deaths 1959 USA H7N7 1 0 1995 UnitedKingdom H7N7 1 0 1997 Hong Kong H5N1 18 6 1998 China H9N2 5 0 1999 HongKong H9N2 2 0 2003 Hong Kong H5N1 2 1 2003 Netherlands H7N7 89 1 2003Hong Kong H9N2 1 0 2004 Vietnam H5N1 33 25 2004 Thailand H5N1 17 12 2004Canada H7N3 2 0

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1. A method for producing an influenza virus and/or influenza viralproteins for use as a vaccine, said method comprising: providing a cellwith at least a sequence encoding at least one gene product of anadenoviral E1 gene or a functional derivative of said adenoviral E1gene; infecting said cell with an influenza virus; culturing said cellin a suitable medium and allowing for expression of said influenzavirus; and harvesting said influenza virus from said suitable mediumand/or said cell, wherein said influenza virus is an influenza virusstrain that is associated with a pandemic outbreak, or has the potentialto be associated with a pandemic outbreak.
 2. The method according toclaim 1, wherein said influenza virus strain is selected from the groupconsisting of H5N1, H5N2, H5N8, H5N9, H7N1, H7N3, H7N4, H7N7, and H9N2.3. The method according to claim 1, wherein said cell is a human primarycell.
 4. The method according to claim 1, wherein said cell isimmortalized by a gene product of said adenoviral E1 gene.
 5. The methodaccording to claim 1, wherein said cell is derived from a humanembryonic retinoblast.
 6. The method according to claim 1, wherein thesequence encoding at least one gene product of the adenoviral E1 gene ispresent in the genome of said human cell.
 7. The method according toclaim 1, wherein said cell does not produce adenoviral structuralproteins.
 8. The method according to claim 1, wherein said cellcomprises no other adenoviral sequences.
 9. The method according toclaim 1, wherein said cell is capable of growing in suspension.
 10. Themethod according to claim 1, wherein said cell is cultured in theabsence of serum.
 11. The method according to claim 1, wherein said cellis a cell as deposited under ECACC no. 96022940 or a derivative thereof.12. The method according to claim 1, further comprising the step ofattenuating, inactivating or disrupting said produced virus.
 13. Themethod according to claim 12, wherein said virus is disrupted, furthercomprising the step of purifying haemagglutinin and neuraminidaseproteins from said influenza virus.
 14. A method for producing aninfluenza vaccine, said method comprising the steps of: producinghaemagglutinin and neuraminidase proteins from an influenza virus strainthat is associated with a pandemic outbreak, or has the potential to beassociated with a pandemic outbreak, according to the method of claim13; and mixing said proteins with a therapeutically acceptableexcipient.
 15. A vaccine comprising an influenza virus, produced by themethod of claim
 14. 16. The vaccine of claim 15, further comprising anadjuvant.
 17. The vaccine of claim 16, wherein said adjuvant is aluminumhydroxide, aluminum phosphate, or both aluminum hydroxide and aluminumphosphate.
 18. A process for producing haemagglutinin proteins andneuraminidase proteins of an influenza virus strain selected from thegroup of influenza virus strains consisting of H5N1, H5N2, H5N8, H5N9,H7N1, H7N3, H7N4, H7N7, and H9N2, said process comprising: providing acell, wherein said cell is a human cell that has been immortalized by atleast one gene product of an adenoviral E1 gene; infecting said cellwith an influenza virus selected from the group of influenza virusstrains consisting of H5N1, H5N2, H5N8, H5N9, H7N1, H7N3, H7N4, H7N7,and H9N2; culturing said infected cell in a medium suitable forculturing the cell, expressing said influenza virus in said infectedcell; disrupting the expressed influenza virus, and isolatinghaemagglutinin proteins and neuraminidase proteins from the disruptedinfluenza virus.
 19. A composition comprising: a sufficient amount ofisolated haemagglutinin proteins and neuraminidase proteins of aninfluenza strain to produce an immunological response in a mammalian oravian subject, said influenza strain selected from the group consistingof H5N1, H5N2, H5N8, H5N9, H7N1, H7N3, H7N4, H7N7, and H9N2, whereinsaid isolated haemagglutinin proteins and neuraminidase proteins areproduced by the process of claim
 18. 20. The vaccine of claim 19 furthercomprising an adjuvant.